The present invention relates to a method for a high-concentration incubation of nitrifying bacteria (marine nitrifying bacteria and freshwater nitrifying bacteria) or denitrifying bacteria (marine denitrifying bacteria and freshwater denitrifying bacteria) using activated sludge as a raw material, to an incubation promoter used therefor and to a method for a weight reducing treatment of the activated sludge.
With regard to a method for the elimination of pollution by nitrogen, particularly by ammonia, in an aqueous system, there are a physicochemical method and a method utilizing organisms.
Examples of the physicochemical method are an ammonia stripping method, a discontinuous point chlorine injection method, a zeolite method and an ion-exchange method.
However, in those methods, there are many problems in view of the efficiency and the secondary environmental pollution due to by-products.
On the other hand, with regard to a method utilizing organisms, it has been well known that a method by treating with microorganism using nitrifying bacteria (ammonia oxidizing bacteria and nitrite oxidizing bacteria) and denitrifying bacteria (nitrate oxidizing bacteria) is most useful. Harmful residual nitrogen in an aqueous system is accumulated as nitrate ion as a result of action of nitrifying bacteria under aerobic environment, reduced to safe nitrogen gas by denitrifying bacteria under anaerobic environment and exhausted into air.
However, nitrifying bacteria have a slow growing rate and do not live by means of formation of colonies and, therefore, there has been no report on the success in a high-concentration incubation in a large quantity in an industrial scale up to the present time where more than one hundred years have passed since their presence was confirmed.
Thus, the conventional incubating method is in a small scale of a test tube level with an object of pure culture and, in the case of incubation of about two months, the result is that the medium in a flask is not suspended whereby it is not an incubating method which can be applied in industry.
To be more specific, when nitrification begins in the incubation of nitrifying bacteria, pH lowers and, until now, no method for an effective increase in the pH has been known. On the other hand, carbon sources decrease as a result of nitrification and, until now, carbon dioxide has been used for supplying the carbon source. Although it is of course possible to prevent the exhaustion of carbon sources when carbon dioxide is supplied, the above-mentioned lowering of the pH further proceeds by that whereupon activity of the nitrifying bacteria stops and that is a limit of the growth of the bacteria.
(1) Name of the Depository Institution: Patent Microorganism Depository Center, National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI
Address: Postal Code 305-0046
1-1-3 Higashi, Tsukuba-shi, Ibaraki-ken, Japan (Telephone: 0298 54 6029)
(2) Deposited Date: Apr. 27, 2000
(3) Accession Number: FERM BP-7150
The present invention relates to a method for the incubation of nitrifying bacteria in a high concentration which are contained in a few amounts in activated sludge such as sewage sludge and excrement sludge and is a method for a high-concentration incubation of nitrifying bacteria which is characterized in that the above-mentioned activated sludge is subjected to nitrification and acclimation culture by a liquid containing NH4xe2x80x94N for a predetermined period under such a condition that that dissolved oxygen is not less than 2 mg/liter, pH is 7.0-9.0 and temperature is 20-40xc2x0 C. and, at the same time, the pH which is apt to tend to an acid side during the process of acclimation culture is constantly maintained within the above-mentioned range by addition of an incubation promoter comprising a mixture of sodium carbonate and sodium hydrogen carbonate whereby the nitrifying bacteria contained in the above-mentioned sludge are subjected to acclimation culture and are accumulated.
The present further relates to a method for the incubation of denitrifying bacteria in a high concentration which are contained in a few amounts in activated sludge such as sewage sludge and excrement sludge and is a method for a high-concentration incubation of denitrifying bacteria which is characterized in that the above-mentioned activated sludge is subjected to denitrification and acclimation culture by a liquid containing NO3xe2x80x94N for a predetermined period under such a condition that dissolved oxygen is not more than 2 mg/liter, pH is 6.0-9.0, temperature is 10-40xc2x0 C. and ROH (R is CH3xe2x80x94 and/or C2H5xe2x80x94) is present as an external carbon source whereby the denitrifying bacteria contained in the above-mentioned activated sludge are subjected to acclimation culture and are accumulated.
In accordance with the present invention, it is now possible to incubate the nitrifying bacteria or the denitrifying bacteria in a large quantity and also in a high concentration.
Examples of the activated sludge used in the present invention are sewage sludge and excrement sludge. They may be either subjected to a diluting treatment with fresh water or subjected to a diluting treatment with sea water but, when nitrifying bacteria or denitrifying bacteria are incubated using a sludge diluted with sea water as a raw material, marine nitrifying bacteria and marine denitrifying bacteria which are of scarcity values can be obtained in large quantities and, therefore, it is appropriate to use an activated sludge which is diluted with sea water.
To be more precise, in natural sea water, there are marine nitrifying bacteria which are believed to have higher resistance to salt than freshwater nitrifying bacteria but, since their amount is very small and their separation in a pure state is difficult, studies thereof are in the rear of freshwater nitrifying bacteria. However, in accordance with the incubating method of the present invention, the activated sludge which is diluted with sea water as mentioned already is used whereby it is possible to prepare the marine nitrifying bacteria of a high concentration. The marine nitrifying bacteria are equipped with multi-layered cell walls and have a strong resistance to changes in osmotic pressure and to various chemical substances which inhibit the growth.
In the incubation of nitrifying bacteria contained in activated sludge, the said activated sludge is subjected to nitrification and acclimation culture for a predetermined period (for example, for one month, two months or three months) using waste liquid in sludge treatment such as dehydrated filtrate of sludge and (anaerobic) digested eluate and, since the nitrification and acclimation culture are to be carried out aerobically, it is necessary to make the dissolved oxygen (DO) at that time 2 mg/liter or more. However, it has been firstly found by the experiment at this time that, when the dissolved oxygen concentration is made too high, the growing speed rather tends to lower. That will be illustrated in detail as hereunder.
The more the dissolved oxygen, the quicker the nitrifying speed by the nitrifying bacteria. Therefore, it has been believed that the more the dissolved oxygen, the quicker the nitrification and the acclimation culture as well but, quite unexpectedly, it has been found now that speed of the acclimation culture and the accumulation lowers when the dissolved oxygen (DO) is more than about 5 mg/liter. Incidentally, it is most preferred that the dissolved oxygen (DO) concentration is 2-4 mg/liter.
It is also necessary that pH is 7.0-9.0 and (,especially when activated sludge diluted with sea water is used,) the pH is preferably 7.5-8.5 and, more preferably, 7.5-7.8.
With regard to the temperature for incubation, growing speed is high when it is within a range of 20-40xc2x0 C. and the range of 25-35xc2x0 C. is more preferred.
Incidentally, during the process of incubation, alkalinity lowers when pH lowers. Thus, oxidation of NH4+ to NO2xe2x88x92 by the ammonia oxidizing bacteria and oxidation of NO2xe2x88x92 to NO3xe2x88x92 by the nitrite oxidizing bacteria are expressed by the following two formulae (A) and (B). Incidentally, the formula (C) is a formula in terms of the nitrifying bacteria as a whole.
(Ammonia oxidizing bacteria)
NH4++1.5O2xe2x86x92NO2xe2x88x92+H2O+2H+xe2x80x83xe2x80x83(A)
(Nitrite oxidizing bacteria)
NO2xe2x88x92+0.5O2xe2x86x92NO3xe2x88x92xe2x80x83xe2x80x83(B)
(Mixed system)
NH4++2.0O2xe2x86x92NO3xe2x88x92+H2O+2H+xe2x80x83xe2x80x83(C)
From those formulae, it is noted that 4.57 mg O2/mg NH4xe2x80x94N of oxygen are needed for oxidation of NH4xe2x80x94N to NO3xe2x80x94N and that, since hydrogen ion is released as the nitrification reaction proceeds, the alkalinity decreases when the pH of the incubating system lowers. Since the incubating rate decreases as the pH lowers, activity of the microorganism stops as same as in the conventional methods unless the pH is kept at a predetermined value using a buffer or the like.
Accordingly, in the present invention, with a belief that a substance which is a mixture of nonhydride and hydride to give a buffer action is suitable for adjusting the pH which tends to an acid side during the incubating process, we have repeatedly carried out tries-and-errors using many compounds and, as a result, it has been found to be most appropriate to recover the pH by pouring an incubation promoter comprising a combination of sodium carbonate and sodium hydrogen carbonate thereinto.
It has been known that, generally, synthetic reaction of bacterial cells can be expressed by the following formula.
4CO2+HCO3xe2x88x92+NH4++H2Oxe2x86x92C5H7NO2+5O2
When that is applied to the biochemical reaction formula of the above-mentioned mixed incubation system (C), the result will be nearly as follows.
NH4++1.86O2+1.98HCO3xe2x88x92xe2x86x920.021C5H7NO2+0.98NO3xe2x88x92+1.04H2O+1.88H2CO3
It is apparent from the above formula that a lot of carbon source is necessary for incubation of nitrifying bacteria even as compared with ammonium ion which is an energy substrate.
As mentioned above, when an incubation promoter comprising a combination of sodium carbonate and sodium hydrogen carbonate is supplied, carbon source for the carbon dioxide assimilation can be supplied at the same time. Illustration will be made as hereunder.
When sodium carbonate is solely used, an effect of rising the lowering pH can be well achieved because the said sodium carbonate is strongly alkaline but, since an effect of rising the pH is big, it cannot be used in a large quantity whereby there is an inconvenience for supplying the carbon source sufficiently. On the other hand, when sodium hydrogen carbonate is solely used, there is no problem in terms of supplying the inorganic carbon source but, in terms of keeping the pH, a large quantity is to be supplied and that is not preferred.
In view of the merit and the disadvantage as such, a mixture of sodium carbonate and sodium hydrogen carbonate is advantageously utilized. When an aqueous solution of those compounds is used, it is now possible that the pH which gradually lowers is kept constant and, at the same time, inorganic carbon source for the carbon dioxide assimilation for an organism can be effectively supplied.
With regard to the compounding ratio of sodium carbonate to sodium hydrogen carbonate in the above-mentioned mixture, the molar ratio of sodium carbonate to sodium hydrogen carbonate is preferably 4-7:4-8 and, to be more specific, a mixed aqueous solution of 0.4-0.7 (mol/liter) of sodium carbonate and 0.4-0.8 (mol/liter) of sodium hydrogen carbonate is effective.
Incidentally, monitoring of the pH of the incubation system maybe carried out either continuously or intermittently. Although it is preferred to utilize a continuous pH monitoring apparatus such as a pH controller, the present invention is not limited thereto but it is also possible to carry out by means of a manual work utilizing a pH indicator such as Phenol Red.
Concentration of ammonia in a solution containing NH4xe2x80x94N is preferably from 100 mg/liter to 300 mg/liter and, more preferably, it is not more than 200 mg/liter. Ammonia is an energy source when ammonia oxidizing bacteria which are chemoautorophic bacteria grow by carrying out the carbon dioxide assimilation but, if it is present too much, that may rather inhibit the growth and the proliferation in some cases. In addition, the nitrite oxidizing bacteria which are contained in activated sludge like ammonia oxidizing bacteria are the bacteria which successively oxidize nitrous acid produced by the ammonia oxidizing bacteria but, due to such a property, they are weak to high concentrations of nitrous acid and, therefore, the initial concentration of ammonia is not able to made too high. Accordingly, when the concentration of ammonia exceeds 300 mg/liter, it is suitable to appropriately dilute it with sea water or fresh water.
Incidentally, as a liquid containing NH4xe2x80x94N, it is preferred to use a waste liquid in sludge treatment such as dehydrated filtrate of sludge and digested eluate produced in a water treatment plant.
When activated sludge is subjected to nitrification and acclimation culture using a waste liquid in a sludge treatment such as dehydrated filtrate of sludge and digested eluate under the above-mentioned incubating conditions, the nitrifying bacteria slightly contained in the said activated sludge can be incubated in a high concentration. In addition to that, it is possible according to the present invention to reduce the activated sludge to an extent of one-third to one-fourth within two months and also to prepare nitrified sludge having a high specific gravity.
In the meanwhile, it has been said that activated sludge contains about 0.35% of nitrifying bacteria. When such an active sludge is used as a raw material and subjected to acclimation culture and accumulated for about two months by a liquid containing NH4xe2x80x94N, amount of the nitrifying bacteria in the said activated sludge increases to an extent of about ten-fold (3.5%). During that process, other sundry bacteria die due to cannibalism because no nutritive source (feed) is provided from outside. As a result thereof, amount of the activated sludge reduces (volume is reduced).
When most of the sundry bacteria die, they become a hardly degradable organic matter having a big specific gravity called xe2x80x9cgranulexe2x80x9d and the nitrifying bacteria attach around it as a nucleus. Such a hardly degradable organic matter to which the nitrifying bacteria are attached is sedimented in the incubating system due to its high specific gravity. In incubating the nitrifying bacteria in a high concentration, the good sedimentation property as mentioned above is essential. Thus, generally speaking, nitrifying bacteria have a light specific gravity and are floated in the pure culture. Therefore, there is a big possibility that nitrifying bacteria are flown out from the incubating system whereby a high-concentration incubation cannot be expected. Accordingly, in a high-concentration incubation, production of the above-mentioned nucleus (the hardly degradable organic matter) is essential and generation of nucleus is not noted in pure culture of the nitrifying bacteria but is noted only when the activated sludge is used as a raw material.
Incubation of denitrifying bacteria contained in activated sludge is carried out by subjecting the said activated sludge to a denitrifying accumulation culture by a liquid containing NO3xe2x80x94N for a predetermined period (such as for one month, two months or three months).
A denitrifying reaction is a reaction where an organic energy source (which will be mentioned later) as a hydrogen donor is oxidized using molecular oxygen in NO3xe2x88x92 in a liquid containing NO3xe2x80x94N. Thus, a denitrifying reaction is an oxidation reaction of an organic substance (AH2) in which NO3xe2x88x92 is a final hydrogen acceptor instead of oxygen and is expressed by the following formula (a simplified formula).
NO3xe2x88x92+AH2xe2x86x92A+H2O+N2
Accordingly, this denitrifying acclimation culture is to be carried out anaerobically and, therefore, the dissolved oxygen (DO) is to be not more than 2 mg/liter, preferably not more than 1 mg/liter or, more preferably, not more than 0.5 mg/liter (in other words, it is necessary to prepare a condition whereby a nitrate respiration is able to take place).
The pH may be within a range of 6.0-9.0 and, rather than otherwise, an alkaline side is preferred. To be more specific, the pH is preferably 6.5-8.5, more preferably 7.0-8.5 and, still more preferably, 7.5-8.5.
With regard to the incubating temperature, activity suddenly lowers when it is lower than 10xc2x0 C. and, therefore, the said temperature is to be 10-40xc2x0 C., preferably 15-30xc2x0 C. and, more preferably, 25-30xc2x0 C.
With regard to the liquid containing NO3xe2x80x94N, it is possible to use a nitrified and acclimation-cultured liquid which is produced when incubation of nitrifying bacteria is carried out using the activated sludge (refer to the already-mentioned passage).
As mentioned above, an organic matter is to be supplemented from outside as a hydrogen donor and also as a carbon source for the cell synthesis. With regard to such an organic matter from outside, the use of methanol is preferred because it gives quicker growth speed and is less expensive and easily available. Incidentally, it is also possible to use ethanol together with or instead of methanol.
Although there is no particular limitation for the concentration of methanol and/or ethanol to be added, it is preferred that, in terms of methanol, the value of CH3OH (mg/liter)/NO3xe2x80x94N (mg/liter) is 3.0 or more.